Porphyrin containing lactic acid bacterial cells and use thereof

ABSTRACT

Culturally modified lactic acid bacterial cells containing a porphyrin compound and their use in a novel method of reducing the oxygen content in a food and feed product or starting material is provided and means of improving the shelf life and/or quality of such products by using the culturally modified bacterial cells. Such culturally modified cells are useful in the manufacturing of a food and a feed product and for the manufacturing of metabolites produced by modified cells.

FIELD OF INVENTION

[0001] The present invention relates to the field of lactic acidbacterial starter cultures and in particular to culturally modifiedlactic acid bacterial cells containing a porphyrin compound. Morespecifically, the invention provides a novel method of reducing theoxygen content in a food and feed product or starting material thereforand means of improving the shelf life and/or quality of such products byusing the culturally modified bacterial cells. Thus, such cells areuseful in the manufacturing and preservation of food and feed products.

TECHNICAL BACKGROUND AND PRIOR ART

[0002] Lactic acid bacteria are used extensively in the food and feedindustry in the manufacturing of fermented products including most dairyproducts such as cheese, yoghurt and butter, meat products; bakeryproducts; wine or vegetable products. When used for such purposes,cultures of lactic acid bacteria are generally referred to as startercultures and they impart specific features to various fermented productsby performing a number of metabolic and other functions herein.

[0003] In the present context, the expression “lactic acid bacteria”designates a group of Gram positive, catalase negative, non-motile,microaerophilic or anaerobic bacteria which ferment sugar with theproduction of acids including lactic acid as the predominantly producedacid, acetic acid, formic acid and propionic acid. The industrially mostuseful lactic acid bacteria are found among Lactococcus species,Lactobacillus species, Streptococcus species, Enterococcus species,Leuconostoc species, Oenococcus species and Pediococcus species.

[0004] When lactic acid bacteria are cultivated in a medium like milk orany other starting material in the manufacturing of food and feedproducts, the medium becomes acidified as a natural consequence of thegrowth and metabolic activity of the lactic acid bacterial startercultures. In addition to the production of lactic acid/lactate fromcitrate, lactose or other sugars several other metabolites such as e.g.acetaldehyde, α-acetolactate, acetoin, acetate, ethanol, carbon dioxide,diacetyl and 2,3-butylene glycol (butanediol) are produced during thegrowth of the lactic acid bacteria.

[0005] Generally, the growth rate and the metabolic activity of lacticacid bacterial starter cultures can be controlled by selectingappropriate growth conditions for the strains of the specific starterculture used such as appropriate growth temperature, oxygen tension andcontent of nutrients.

[0006] Although milk is generally an ideal medium for the growth oflactic acid bacteria, a high content of oxygen in the milk affects thegrowth of the bacteria adversely and it is known in the dairy industrythat a reduction of the oxygen content of the milk raw material mayresult in a more rapid growth of the added bacteria which in turnresults in a more rapid acidification of the inoculated milk. Currently,such a reduction of the oxygen content is carried out by heating themilk in open systems, by deaerating the milk in vacuum or by a spargingtreatments. Alternative means of reducing the oxygen content include theaddition of oxygen scavenging compounds or the use of mixed culturescomprising two or more lactic acid bacterial species, at least one ofwhich is less sensitive to oxygen.

[0007] WO 98/54337 discloses a method of enhancing the growth rate oflactic acid bacteria by cultivating the lactic acid bacteria inassociation with a metabolically engineered lactic acid bacterial helperorganism which has a defect in its pyruvate metabolism, resulting in anincreased oxygen consumption by the helper organism. However, thismethod of reducing the oxygen content in a medium is limited to the useof specific modified lactic acid bacterial strains and thus, there is aneed to find a biological method of oxygen reduction in a food or feedstarting material which does not involve the use of specifically mutatedor metabolically modified lactic acid bacteria.

[0008] As mentioned above, when grown anaerobically lactic acidbacterial cells ferment sugars principally to lactic acid/lactate viapyruvate. NADH produced in the cells during this catabolism isreoxidised via lactate dehydrogenase. Under aerobic conditions, however,NADH can partly become reoxidised by NADH peroxidase and oxidase. Thus,under aerobic conditions pyruvate can be converted into other endproducts than those produced under anaerobic conditions which in turnresults in an increase in biomass yield. Most lactic acid bacteria arecatalase negative when grown in a haeme or haematin free medium. NADHperoxidase may therefore act to remove H₂O₂ produced by aerobic culturesof species unable to form a pseudo-catalase.

[0009] It is known that some lactic acid bacteria can form catalase andcytochromes when the aerobic growth medium is supplemented withhaematin, blood or a haemoprotein. Sijpesteijn (1970) showed that thefermentation of strains of Lactococcus lactis and Leuconostocmesenteroides in the presence of 10 ppm of haemin induced profoundchanges in the aerobic breakdown of glucose by resting cells of bothorganisms. This was observed when cells were cultivated under aerobiccondition and, in the presence of haemin and glucose, were transferredinto a resting cell medium, i.e. a medium wherein the cells are notcapable of growth. Furthermore, an increased O₂ uptake was observed andless lactic acid and more acetic acid and acetoin was produced. It wasshown that cytochromes were formed in these organisms when culturedunder the above culture conditions and that the respiration became moresensitive to KCN. It was suggested by this author that, after growth inthe presence of haemin, a cytochrome-mediated respiration regulated byhaemin was mainly responsible for the oxidation of NADH and that NADHoxidase only played a minor role under these conditions.

[0010] In a study on a cytochrome-like system in lactic acid bacteria,Ritchey & Seeley (1976) reported similar results. When grown on ahaematin-containing medium with glucose some strains such as strains ofStreptococcus faecalis or L. lactis, e.g. L. lactis subsp. lactisbiovar. diacetylactis produced cytochromes whereas S. faecium did not.It was stated by these authors that strains like S. faecalis or L.lactis are blocked in the steps of haeme synthesis but possess thegenetic determinants to establish a membrane-bound cytochrome electrontransport chain under appropriate condition.

[0011] However, Kaneko et al. (1990) could not observe cytochromes whenculturing a Citr⁺ (i.e. citrate metabolising) strain of L. lactis underaerobic conditions and in the presence of haemin and/or Cu⁺.Furthermore, they did not observe any difference in NADH oxidase,diacetyl reductase or lactate dehydrogenase activity when culturing thatstrain under these culture conditions. However, they observed anincrease in the production of diacetyl and acetoin due to an activationof diacetyl synthase by haemin and/or Cu⁺. Japanese Patent ApplicationJP 04-36180 and EP 430 406, both filed in the name of Meiji MilkProduction Co. Ltd., proposed using these culture conditions to improvethe production of diacetyl and acetoin by using the Citr⁺ strain of L.lactis.

[0012] The above studies all show that the addition of haemin orhaematin to the fermentation medium under aerobic conditions results inchanges in the aerobic breakdown of glucose and that a possiblecytochrome dependent aerobic respiration is induced in strains culturedunder the above conditions. However, the above documents do not addressthe problem of reducing the oxygen content of the milk raw material orany other starting material in order to increase the growth of the addedbacteria of a starter culture. Although an increased O₂ uptake inresting cell systems under aerobic conditions in the presence of haeminand glucose has been reported, the ability of such cultured strains tobe used in a starter culture for the manufacturing of a food or feedproduct has not been suggested.

[0013] The present invention is based on the discovery that lactic acidbacterial strains, when cultured or fermented under aerobic conditionsin the presence of haemin and other porphyrin compounds, are capable ofmaintaining their increased oxygen reducing activity/capability wheninoculated into milk or any other media under appropriate conditions andwithout addition of a porphyrin compound. It is thus possible to providea generally applicable biological method for reducing the oxygen contentin milk or any other food or feed starting material, whereby the growthand metabolic activity of lactic acid bacterial starter cultures hereincan be substantially enhanced. It is therefore a primary objective ofthe present invention to provide such a method and culturally modifiedcells, which are useful in such a method.

[0014] Accordingly, these findings have opened up for a novel approachfor providing useful culturally modified lactic acid bacterial startercultures, which approach is based on relatively simple classicalfermentation methods and which does not involve genetic engineering orclassical mutagenesis. From a practical technological point of view thisis advantageous, since the use of genetic engineering or classicalmutagenesis in the construction of new strains is very labour intensiveand costly and the use of genetically modified organisms in foodproduction may give rise to consumer concerns.

SUMMARY OF THE INVENTION

[0015] Accordingly, the present invention provides in a first aspect aculturally modified lactic acid bacterial cell that has, relative to thecell from which it is derived, an increased content of a porphyrincompound.

[0016] In a further aspect there is provided a starter culturecomposition comprising the culturally modified lactic acid bacterialcell according to the invention.

[0017] In another aspect, there is provided a method of reducing theoxygen content in a food or feed product or in a food or feed productstarting material, the method comprising adding to the product or to thestarting material an effective amount of the culturally modified lacticacid bacterial cells according to the invention or the above starterculture composition.

[0018] In yet another aspect, there is provided a method of improvingthe shelf life and/or the quality of an edible product, comprisingadding to the product an effective amount of the culturally modifiedlactic acid bacterial cells according to the invention or the abovestarter culture composition.

[0019] In a still further aspect, the invention pertains to a method ofpreparing a fermented food or feed product, comprising adding aneffective amount of the culturally modified lactic acid bacterial cellaccording to the invention or the above composition to a food or feedproduct starting material, wherein the cell or the composition iscapable of fermenting said starting material to obtain the fermentedfood or feed.

[0020] The invention pertains in other aspects to the use of the abovemodified lactic acid bacterial cell or the composition comprising suchcells for the production of a metabolite or for the production of abacteriocin.

DETAILED DISCLOSURE OF THE INVENTION

[0021] It is a primary objective of the present invention to provide agenerally applicable biological method of reducing the oxygen content ina food or feed product or in a starting material for such products.Accordingly, in one aspect there is provided a culturally modifiedlactic acid bacterial cell that has, relative to the cell from which itis derived, an increased content of a porphyrin compound.

[0022] As used herein the expression “culturally modified lactic acidbacterial cell” relates to a cell of a lactic acid bacterium which hasbeen cultured by fermentation in an appropriate nutrient medium in whichan effective amount of at least one porphyrin compound is present. Inthe present context, the expression “an effective amount” means anamount that is sufficient to cause the lactic acid bacterium to becomemodified as defined herein. It will be understood from the discussionabove, that the presence of the porphyrin compound causes the cellscultured under such conditions to have a modified aerobic breakdown ofcarbohydrates, such as lactose, glucose or galactose. After fermentationthe bacterial cells are harvested using conventional procedures andsubsequently used as a starter culture for the inoculation of a milkmedium or any another starting material for food or feed manufacturingwherein the cells are capable of replicating, with or without theaddition of a porphyrin compound to the medium. As used herein, the term“fermentation” refers to a process of propagating or cultivating alactic acid bacterial cell under both aerobic and anaerobic conditions.

[0023] As shown in the below examples, it was possible for the inventorsof the present invention to detect the presence and the amount of aporphyrin compound in cells which have been cultured in the presence ofa porphyrin compound both when analysing the culturally modified cellsafter fermentation or after the cells have been transferred to a mediumwherein the cells are capable of replicating. It was surprising when theinventors observed that the addition of a porphyrin compound to theculture medium results in an increased content of the porphyrin compoundin the culturally modified cells relative to the cells from which theyare derived. It will be appreciated that the expression “relative to thecells from which they are derived” relates to similar lactic acidbacterial cells, which, in contrast to the culturally modified bacterialcells according to the invention, were not cultured in a mediumcontaining a porphyrin compound.

[0024] Thus, in a preferred embodiment, the cell according to theinvention contains at least 0.1 ppm on a dry matter basis of a porphyrincompound, including at least 0.2 ppm, such as at least 0.5 ppm,including at least 1 ppm, e.g. at least 2 ppm, such as at least 5 ppm,including such as 10 ppm, such as at least 20 ppm, e.g. at least 30 ppm,such as at least 40 ppm, e.g. at least 50 ppm, such as at least 60 ppm,e.g. at least 70 ppm, such as at least 80 ppm, e.g. at least 90 ppm,such as at least 100 ppm on a dry matter basis of a porphyin compound.

[0025] “Porphyrin compounds” refers in the present context to cyclictetrapyrrole derivatives, whose structures are derived from that ofporphyrin by substitution at the carbon atoms located at the apices ofthe pyrrole core, with various functional groups. It also refers tocomplexes of said derivatives with a metal atom that forms coordinatebonds with two of the four nitrogens of the porphyrin ring. Thedefinition encompasses also, but is not limited to, uroporphyrins,coproporphyrins, protoporphyrins and haematoporphyrins including theirsalts and esters and their complexes with a metal atom, preferably aniron atom, the dihydrochloride of coproporphyrin I, the tetraethyl esterof coproporphyrin III, the disodium salt of protoporphyrin IX, thedichloride of haematoporphyrin IX, the tetraisopropyl ester or thetetramethyl ester of coproporphyrin, the tetraisopropyl ester or thetetramethyl ester of coproporphyrin III, haematoporphyrin IX,haemoglobin, protoporphyrin IX, the dimethyl ester of protoporphyrin IX,zinc protoporphyrin IX, haematin and cytohaemin. Particularly preferredporphyrin compounds are protoporphyrin IX and its complexes with an ironatom, in particular haeme and haemin. Furthermore, the definitionencompasses various chlorophylls, such as chlorophyll a and chlorophyllb, their derivatives such as chlorophyllins and also their salts andesters, and their complexes with a metal atom, such as an iron, copperor magnesium atom.

[0026] As it is further shown in the below examples, when using theHPLC-MS method the inventors observed in cells grown on a nutritionalmedium containing a porphyrin compound both under anaerobic and aerobicconditions clear peaks in the porphyrin region. No cytochromes weredetected in cells cultured in a medium without haemin. However, it hasbeen found that it is possible to detect cytochromes at a relativelyhigh level in the modified cells after they have been transferred to amedium wherein the cells are capable of replicating. This is a verysurprising finding, as it has not hitherto been shown that lactic acidbacterial cells grown under aerobic conditions in the presence of aporphyrin compound has an increased content of cytochromes and that theculturally modified cells contain cytochromes after inoculation into amedium wherein the cells are capable of replicating. Without intendingto limit the invention in any way, the inventors propose that abacterial cell grown under aerobic conditions may, through the action ofan NADH oxidase, regenerate the required NAD⁺ under oxygen consumption.If a porphyrin compound is present in the culture medium under aerobicconditions the bacterial cells produce cytochromes and, due to thecytochrome dependent respiration, oxygen is reduced to water with theformation of metabolically usable energy, such as ATP and NAD⁺.

[0027] It will be understood that, although the formation of cytochromesin lactic acid bacterial cells is induced by the presence of a porphyrincompound, it is possible to observe cells grown under such conditionshaving, relative to the cells from which they are derived, an increasedcontent of a porphyrin compound without the formation of cytochromes.Thus, it is possible to detect and measure in the lipid membranes of themodified cells according to the invention both free haemin, haemin boundto a non-cytochrome protein and haemin bound to a cytochrome protein toform a complex. In addition, it is possible to observe in the cytoplasmof the modified cells both free cytoplasmic haemin, cytoplasmic haeminbound to a non-cytochrome protein and cytoplasmic haemin bound to acytochrome protein to form a complex.

[0028] In the present context the term “cytochrome” relates to a groupof electron-transporting proteins containing a haeme prosthestic groupand thus to components of the respiratory and photosynthetic electrontransport chains, in which the haeme iron exits in oxidised or reducedstate. The definition encompasses, but is not limited to, cytochromes ofa-, b-, c-, d- or o-types and combinations of these cytochrome types ase.g. mentioned in Wachenfeldt & Hederstedt (1992). It will beunderstood, that the term “the respiratory electron transport chain”refers to either an aerobic respiratory electron transport chainfunctioning with molecular oxygen as terminal electron acceptor, or ananaerobic respiratory electron transport chain functioning with otherterminal electron acceptors than molecular oxygen such as nitrate,sulphate, fumarate or trimethylamine oxide.

[0029] Thus, in a preferred embodiment, the cells according to theinvention contain at least 0.1 ppm on a dry matter basis of acytochrome, including at least 0.2 ppm, such as at least 0.5 ppm,including at least 1 ppm, e.g. at least 2 ppm, such as at least 5 ppm,including such as 10 ppm, such as at least 20 ppm, e.g. at least 30 ppm,such as at least 40 ppm, e.g. at least 50 ppm, such as at least 60 ppm,e.g. at least 70 ppm, such as at least 80 ppm, e.g. at least 90 ppm,such as at least 100 ppm on a dry matter basis of a cytochrome.

[0030] In accordance with the invention, any starter culture organismswhich are of use in the food or feed industry, including the dairyindustry, can be used. Thus, the cells can be selected from a lacticacid bacterial species including Lactococcus spp., Lactobacillus spp.,Leuconostoc spp., Pediococcus spp., Streptococcus spp.,Propionibacterium spp., Bifidobacterium spp. and Oenococcus spp. In aspecific embodiment the cells are of Lactococcus lactis, includingLactococcus lactis subsp. lactis strain CHCC373 deposited under theaccession number DSM12015.

[0031] Although it is a primary objective of the present invention toprovide a generally applicable biological method for reducing the oxygencontent in milk or any other starting material, i.e. without the use ofgenetically engineered or mutated organisms, it will be appreciated thatthe modified bacteria of the invention may also be a previouslygenetically modified strain of one of the above lactic acid bacterialstrains or any other starter culture strain. As used herein theexpression “genetically modified bacterium” is used in the conventionalmeaning of that term i.e. it includes strains obtained by subjecting alactic acid bacterial strain to any conventionally used mutagenizationtreatment including treatment with a chemical mutagen such asethanemethane sulphonate (EMS) or N-methyl-N′-nitro-N-nitroguanidine(NTG), UV light or to spontaneously occurring mutants. Futhermore, it ispossible to provide the genetically modified bacteria by randommutagenesis or by selection of spontaneously occurring mutants, i.e.without the use of recombinant DNA-technology and it is envisaged thatmutants of lactic acid bacteria can be provided by such technologyincluding site-directed mutagenesis and PCR techniques and other invitro or in vivo modifications of specific DNA sequences once suchsequences have been identified and isolated.

[0032] It was, as mentioned above, very surprising, when the inventorsfound that cells of lactic acid bacterial strains, when they arecultured or fermented under aerobic conditions in the presence of aporphyrin compound, become capable of maintaining their increasedcapability to reduce oxygen, obtained during the fermentation, when theyare inoculated into milk or any other non-porphyrin-containing orlow-porphyrin-containing starter material under appropriate conditions.However, it is also possible to observe this ability when suchculturally modified cells are inoculated in a medium wherein a porphyrincompound is added. Furthermore, the inventors were able to show that theaerobic breakdown of lactose resulted in an increased O₂ uptake.

[0033] Thus, in a preferred embodiment of the present invention, theculturally modified lactic acid bacterial cells are cells which, whenthey are inoculated at a concentration of about 10⁷ cells/ml into lowpasteurised skimmed milk having 8 ppm of dissolved oxygen and leavingthe milk to stand for about 2 hours at a temperature of about 30° C.consumes at least 25% of the oxygen.

[0034] However, the culturally modified lactic acid bacterial cellsaccording to the invention may be particularly useful when the cellsunder the above conditions consumes at least 30% of the oxygen presentin the milk, including at least 40%, such as at least 50%, e.g. at least60%, such as at least 70%, e.g. at least 80%, such as at least 90%, e.g.at least 95% of the dissolved oxygen.

[0035] It has been found that a culturally modified lactic acidbacterial cell according to the invention has, relative to the cell fromwhich it is derived, an altered NADH oxidase, i.e. NOX activity, and/orlactate dehydrogenase (LDH) activity. As explained above, cells grownunder aerobic condition may be capable of regenerating the requiredenergy from other systems induced during aerobic fermentation andmaintained during the inoculation of the cells into milk or any othernon-porphyrin-containing or low-porphyrin-containing starter material.Thus, in preferred embodiments of the present invention the culturallymodified cell is a cell which, relative to the cell from which it isderived, has a decreased NOX activity and/or a decreased LDH activity.It will be understood that the NOX enzyme, which is encoded by the noxgene, is one example of an H₂O forming NADH oxidase. This enzymeregenerates two equivalents of NAD⁺ under consumption of molecularoxygen. Further examples of NADH oxidases which could be present in acell according to the invention are non-haeme flavoproteins where twotypes have been reported, one which catalyses the reduction of O₂ toH₂O₂, the other one the reduction of O₂ to H₂O.

[0036] Accordingly, in further embodiments of the present invention, theculturally modified cell has a NOX activity which is decreased by atleast 10%, 20%, 30%, 40%, 50%, 60%, 75%, or 95% relative to the cellfrom which it is derived. In an interesting embodiment, the modifiedcell has a NOX activity which is decreased by about 100% relative to thecell from which it is derived, i.e. the NOX activity is essentiallyabsent in that modified cell.

[0037] In still further embodiments, the culturally modified cell has aLDH activity which is decreased by at least 10%, including at least 20%,30%, 40%, 50%, 60%, 75% or 95% relative to the cell from which it isderived. In an useful embodiment, the modified cell has a LDH activitywhich is decreased by about 100% relative to the cell from which it isderived, i.e. the LDH activity is essentially absent in that modifiedcell.

[0038] The culturally modified lactic acid bacterial cells according tothe invention are useful as starter cultures in the production of foodand feed products. Accordingly, in a further aspect, the inventionrelates to a starter culture composition comprising the culturallymodified lactic acid bacterial cell according to the invention having,relative to the cell from which it is derived, an increased content of aporphyrin compound.

[0039] It is convenient to provide the starter culture compositionaccording to the invention as a starter culture concentrate both whenused in food and feed production or for the production of metabolitesthat are generated by the starter culture strains. Typically, such aconcentrate contains cells of the starter culture organisms as anon-concentrated fermentate of the respective starter culture strain(s)or in a concentrated form. Accordingly, the starter culture compositionof the invention may have a content of viable cells (colony formingunits, CFUs) which is at least 10⁴ CFU/g including at least 10⁵ CFU/g,such as at least 10⁶ CFU/g, e.g. at least 10⁷ CFU/g, 10⁸ CFU/g, 10⁹CFU/g, 10¹⁰ CFU/g or 10¹¹ CFU/g of the composition.

[0040] The starter culture composition according to the invention can beprovided as a liquid, frozen or dried, such as e.g. freeze-dried orspray-dried, starter culture composition.

[0041] As it is normal in lactic acid bacterial fermentation processesto apply mixed cultures of lactic acid bacteria, the compositionaccording to the invention comprises in certain embodiments amultiplicity of strains either belonging to the same species orbelonging to different species. Accordingly, in a further embodiment,the starter culture composition comprises cells of two or more differentlactic acid bacterial strains. A typical example of such a usefulcombination of lactic acid bacterial cells in a starter culturecomposition is a mixture of the culturally modified lactic acidbacterial cell according to the invention and one or more Lactococcusspp. such as L. lactis subsp. lactis or L. lactis subsp. lactis biovar.diacetylactis or Leuconostoc spp. Such a mixed culture can be used inthe manufacturing of fermented milk products such as buttermilk andcheese. Another example is a mixture of Streptococcus thermophilus andLactobacillus delbrueckii subsp. bulgaricus.

[0042] In one embodiment, the composition according to the invention isa composition which further comprises at least one component thatenhances the viability of the bacterial cell during storage, including abacterial nutrient, a vitamin and/or a cryoprotectant. In the case of acomposition subjected to a freezing step, a suitable cryoprotectant isselected from the group consisting of glucose, lactose, raffinose,sucrose, trehalose, adonitol, glycerol, mannitol, methanol, polyethyleneglycol, propylene glycol, ribitol, alginate, bovine serum albumin,carnitine, citrate, cysteine, dextran, dimethyl sulphoxide, sodiumglutamate, glycine betaine, glycogen, hypotaurine, peptone, polyvinylpyrrolidine and taurine. The cryoprotectant used is advantageouslyselected from alginate, glycerol, glycine betaine, trehalose andsucrose.

[0043] In accordance with the invention, there is also provided a methodof reducing the oxygen content in a food or feed product or in a food orfeed product starting material, which method comprises adding to theproduct or to the starting material an effective amount, e.g. in theform of a suspension, of the culturally modified lactic acid bacterialcells according to the invention or a starter culture compositionaccording to the invention.

[0044] Industrial production of edible products typically includesprocess steps such as mixing, pumping or cooling whereby the degree ofoxygen saturation of the edible product is increased and, as a result,the edible product starting material may have a relatively high initialoxygen content (high degree of oxygen saturation) which is unfavourablefor lactic acid bacterial starter cultures. It has now surprisingly beenfound that when a suspension of the culturally modified lactic acidbacterial cells according to the invention or a starter culturecomposition of this invention is cultivated in an edible food or feedproduct starting material having an initial degree of oxygen saturationof at least 8%, e.g. 10% or higher such as 20% or higher, the starterculture is capable of reducing the oxygen content in the startingmaterial to a content, which is favourable for lactic acid bacterialstarter cultures.

[0045] In the present context, the expression “reducing the oxygencontent” refers to the capability of the suspension of the culturallymodified lactic acid bacterial cells containing a porphyrin compound orthe starter culture composition according to the invention to reduce theinitial content of the oxygen in a medium not supplemented withporphyrin compound.

[0046] In useful embodiments of the method of the present invention theculturally modified lactic acid bacterial cells or the starter culturecomposition according to the invention is one, which is capable ofreducing the amount of oxygen present in the medium by at least 1% perhour including by at least 10% per hour, such as by at least 20% perhour, e.g. by at least 30% per hour. The reduction may even be by atleast 40% per hour including by at least 50% per hour, such as by atleast 60% per hour, e.g. by at least 70% per hour, such as by at least80% or by at least 90% per hour.

[0047] In a specific embodiment of the method according to the inventionthe lactic acid bacterial starter culture is a mixed strain culturecomprising at least two strains of lactic acid bacteria. Examples ofsuch mixed strain cultures are described above. Thus, in particularlypreferred embodiments of the invention the culturally modified cells,when inoculated as a mixed culture comprising cells of at least onefurther culture strain which was not cultivated under aerobic conditionsin the presence of a porphyrin compound, are capable of enhancing thegrowth rate of that further lactic acid bacterial culture strain. Growthconditions, which are in all respects optimal for all strains of suchlactic acid bacterial mixed strain cultures, may not be found.Therefore, the metabolic activity of a mixed strain culture may becontrolled selectively by choosing a temperature which favours anincreased production of desired metabolites by one or more strains, butwhich on the other hand may result in a decreased production of othermetabolites by other strains. However, the overall result of cultivatinga lactic acid bacterial mixed strain culture with the culturallymodified cell according to the invention as compared to the lactic acidbacterial mixed strain culture being cultivated alone is an increasednumber of cells, an increased production of one or more metabolites,including acids and aroma compounds and/or a decreased production of oneor more metabolites.

[0048] Evidently, the above-mentioned enhanced production of acids ofthe lactic acid bacterial starter culture will result in a pH decreaseof the medium inoculated with the culturally modified cells of theinvention which exceeds that obtained in the same medium inoculated withthe starter culture alone. The difference in pH of the medium inoculatedwith the starter culture alone and the medium inoculated with thestarter culture in association with a suspension of the cell accordingto the invention is referred to herein as ΔpH. In useful embodiments ofthe present method the enhanced acid production results in a ΔpH of atleast 0.05 after 3 hours or more of cultivation, such as a ΔpH of atleast 0.1 after 3 hours or more of cultivation, e.g. a ΔpH of at least0.5 after 3 hours or more of cultivation, such as a ΔpH of at least 0.8after 3 hours or more of cultivation, e.g. a ΔpH of at least 1.0 after 3hours or more of cultivation.

[0049] In preferred embodiments of the present invention the ratiobetween culturally modified cells and non-modified lactic acid bacterialstarter culture cells is in the range of 1000:1 to 1:1000 such as 500:1to 1:500, e.g. 100:1 to 1:100, such as in the range of 50:1 to 1:50, e.gin the range of 20:1 to 1:20, such as in the range of 10:1 to 1:10 or inthe range of 5:1 to 1:5 such as in the range of 2:1 to 1:2.

[0050] In further embodiments of the present method the amount ofculturally modified cells is in the range of 10³ to 10¹² CFU per gstarting material. Accordingly, the modified cells are added in amountswhich result in a number of viable cells which is at least 10³ colonyforming units (CFU) per g of the edible product starting material, suchas at least 10⁴ CFU/g including at least 10⁵ CFU/g, such as at least 10⁶CFU/g, e.g. at least 10⁷ CFU/g, such as at least 10⁸ CFU/g, e.g at least10⁹ CFU/g, such as at least 10¹⁰ CFU/g, e.g. at least 10¹¹ CFU/g of thestarting material.

[0051] In useful embodiments, the starting material is a startingmaterial for an edible food product including milk, a vegetablematerial, a meat product, a must, a fruit juice, a wine, a dough and abatter. As used herein, the term “milk” is intended to mean any type ofmilk or milk component including e.g. cow's milk, human milk, buffalomilk, goat's milk, sheep's milk, dairy products made from such milk, orwhey.

[0052] In further embodiments, the starting material is a startingmaterial for an animal feed such as silage e.g. grass, cereal material,peas, alfalfa or sugar-beet leaf, where bacterial cultures areinoculated in the feed crop to be ensiled in order to obtain apreservation hereof, or in protein rich animal waste products such asslaughtering offal and fish offal, also with the aims of preserving thisoffal for animal feeding purposes.

[0053] Yet another significant embodiment of the method according to thepresent invention is where the culturally modified bacterial cells isderived from a bacterial culture generally referred to as a probioticculture. By the term “probiotic” is in the present context understood amicrobial culture which, when ingested in the form of viable cells byhumans or animals, confers an improved health condition, e.g. bysuppressing harmful microorganisms in the gastrointestinal tract, byenhancing the immune system or by contributing to the digestion ofnutrients.

[0054] As it is described above, the culturally modified lactic acidbacterial cells containing a porphyrin compound or the starter culturecomposition according to the invention are capable of reducing theamount of oxygen in a medium. It has been found that such strains canimprove the shelf life of edible products, due to their oxygen reducingability.

[0055] Accordingly, it is another objective of the invention to providea method of improving the shelf life and/or the quality of an edibleproduct, the method comprising adding to the product an effective amountof a suspension of the culturally modified lactic acid bacterial cellsaccording to the invention or a starter culture composition according tothe invention. As used herein the term “shelf life” indicates the periodof time in which the edible product is acceptable for consumption.

[0056] The above shelf life improving effect can be obtained in avariety of edible product components or ingredients such as milkincluding non-pasteurised (raw) milk, meat, flour dough, wine and plantmaterials, such as vegetables, fruits or the above mentioned foddercrops.

[0057] It is also an objective of the present invention to provide amethod of preparing a fermented food or feed product based on the use ofthe culturally modified lactic acid bacterial strain according to theinvention. In its broadest aspect, such method comprises that aneffective amount of the culturally modified lactic acid bacterial cellsaccording to the invention or a composition comprising the modifiedcells are added to a food or feed product starting material, wherein thecells or the composition is capable of fermenting said starting materialto obtain the fermented food or feed. It will be appreciated that insuch a method one or more strains of non-metabolically modified lacticacid bacteria can be used in addition to the modified lactic acidbacteria.

[0058] Useful food product starting materials include any material whichis conventionally subjected to a lactic acid bacterial fermentation stepsuch as milk, vegetable materials, meat products, fruit juices, must,wines, doughs and batters. In further embodiments, the resultingfermented food product in the method of the invention is a dairy productsuch as cheese and buttermilk. In still further embodiments, thestarting material is a starting material for an animal feed such assilage e.g. grass, cereal material, peas, alfalfa or sugarbeet leaf.

[0059] It is yet another objective of the invention to provide the useof the culturally modified lactic acid bacterial cells of the inventionor the composition comprising such cells for the production of ametabolite produced by the cell or the composition. In the presentcontext “produced by the cell or the composition” implies that themetabolite can be one that is naturally produced by the cells or thatthe metabolite is produced recombinantly by the modified cells. In analternative embodiment, the production of the metabolite is achieved byco-cultivating a modified cell of the invention with at least onenon-modified organism capable of producing the metabolite. Typicalexamples of metabolites that is produced by the cells include lacticacid, acetaldehyde, α-acetolactate, acetoin, acetate, ethanol, diacetyland 2,3-butylene glycol.

[0060] Additionally, the cells of the invention and compositionscomprising such cells are useful for the production of bacteriocinsproduced naturally or recombinantly by the cells or another,non-modified cell that is co-cultivated with the modified cell of theinvention, such as e.g. nisin, reuterin and pediocin.

[0061] The invention will now be described in further details in thefollowing non-limiting examples and the drawings wherein

[0062]FIG. 1a shows the chromatogram of a haemin (ferriprotoporphyrin IXchloride) standard 10 μg/ml. Haemin is eluting after 33.3 min;

[0063]FIG. 1b shows the spectrum of the haemin peak after 33.3 min. Themolecular ion of haemin is seen at m/z 616.3 (m/z=mass/charge;cps=counts per second);

[0064]FIG. 2a shows the chromatogram of the cell debris sample offermentation A of the below Experiment 1;

[0065]FIG. 2b shows the spectrum of the peak at time 33.3 min in FIG.2a. The molecular ion of haemin is not seen at m/z 616.3;

[0066]FIG. 3a shows the chromatogram of the cell debris sample offermentation B of the below Experiment 1;

[0067]FIG. 3b shows the spectrum of the peak at time 33.3 min in FIG.3a. The molecular ion of haemin is seen at m/z 616.3;

[0068]FIG. 4a shows the chromatogram of the cell debris sample offermentation C of Experiment 1;

[0069]FIG. 4b shows the spectrum of the peak at time 33.3 min in FIG.4a. The molecular ion of haemin is not seen at m/z 616.3;

[0070]FIG. 5a shows the chromatogram of the cell debris sample offermentation D of Experiment 1;

[0071]FIG. 5b shows the spectrum of the peak at time 33.3 min in FIG.5a. The molecular ion of haemin is seen at m/z 616.3;

[0072]FIG. 6a shows the chromatogram of a Cytochrome c standard 100μg/ml. Cytochrome c is eluting after 23.4 min;

[0073]FIG. 6b shows the spectrum of the peak at time 23.4 min in FIG.6a. The molecular ion of ironporphyrin IX is seen at m/z 617.3 as wellas the multiple charged intact protein (m/z 1031; 1124; 1237; 1374;1546; 1766) with an estimated molecular weight of 12356 Da; and

[0074]FIG. 7 shows the CN-Ox (thin line), the CN/Red-CN (dashed line)and the Red-Ox (thick line) spectra for cells harvested fromfermentation E described in Experiment 2 below.

EXAMPLE 1

[0075] A study of the effect of the addition of haemin to thefermentation medium on the enzyme and oxygen content reducing activitiesof a lactic acid bacterium

[0076] The effect of the addition of haemin to the fermentation mediumon the enzyme activity and the oxygen content reducing activity of alactic acid bacterial strain was studied in two experiments, Experiment1 and Experiment 2. Furthermore, the presence of haemin and cytochromesin lactic acid bacterial cells when grown in a medium containing haeminwas studied.

Experiment 1

[0077] 1. Materials and methods

[0078] 1.1 Microorganism

[0079] The wild type strain Lactococcus lactis subsp. lactis CHCC373(from the Chr. Hansen Culture Collection) was applied in thisexperiment. A sample of the strain was deposited in accordance with theBudapest Treaty with the Deutsche Sammiung von Mikroorganismen undZellkulturen (DSMZ), Marscheroder Weg, 1b, D-38124 Braunschweig on Feb.17, 1998 under the Accession No. DSM 12015.

[0080] 1.2 Medium composition

[0081] The fermentation medium had the following composition: Caseinpeptone, 30 g/l; Primatone, 30 g/l; soy peptone, 30 g/l; yeast peptone,15 g/l; MgSO₄, 1.5 g/l; Na-ascorbate, 3 g/l; and lactose, 30 g/l.Antifoam (Dow Corning 1510) was added at a concentration of 0.25 g/l.

[0082] The medium was sterilised by UHT-treatment. The finished mediumhad a pH of 6.5.

[0083] A fresh solution of haemin was prepared as follows: 1 gram ofhaemin (Fluka prod. no. 51280, molecular weight 651.96 g/mol) wasdissolved in 1 ml 6.7 N NH₄OH and water was added to a final volume of 1liter. The solution was subsequently autoclaved at 121° C. for 20 min.The haemin was added to fermentations B and D as described in thefollowing at a final concentration of 10 mg/I.

[0084] 1.3 Fermentation conditions

[0085] A series of four fermentations with the strain CHCC373 wascarried out using the fermentation medium defined above:

[0086] Fermentation A—Anaerobic fermentation

[0087] Fermentation B—Anaerobic fermentation with haemin addition

[0088] Fermentation C—Aerobic fermentation

[0089] Fermentation D—Aerobic fermentation with haemin addition

[0090] The fermentations were inoculated with a concentrated cellsuspension of CHCC373.

[0091] The anaerobic fermentations were run with nitrogen in headspacewhereas the aerobic fermentations were sparged with air at a rate of 0.3liters per minute per liter of fermentation volume (at this level ofaeration, the dissolved oxygen concentration was maintained above 65% ofsaturation level throughout the aerobic fermentations).

[0092] All four fermentations were run at a temperature of 30° C. andwith a headspace pressure of about 2 bar. The cultures were allowed toacidify to pH 6.2. The pH was subsequently maintained at 6.2 bycontrolled addition of 13.4 N NH₄OH.

[0093] Samples of 10 ml were collected throughout the fermentations formeasurements of the optical density using a Hitachi U-1100Spectrophotometer at 600 nm, and subsequent determination of oxygenreducing effect. The samples were stored at −50° C. until analysis.

[0094] When no further base consumption was observed, the respectiveculture was cooled to about 10° C.

[0095] 1.4 Downstream processing

[0096] Following cooling, each of the four fermentation broths A-D wereconcentrated by centrifugation and subsequently frozen as pellets inliquid nitrogen. The frozen pellets were stored at −80° C. until furtheranalysis.

[0097] 1.5 Sample preparation

[0098] About 10 g of frozen pellets from each fermentation was weighedout accurately. These pellets were washed twice in cold 40 ml 0.05 MNa-phosphate buffer (pH 6.1). Following each step of washing thesuspensions were centrifuged at 9,000 rpm (Sorvall centrifuge with SS-34rotor) for 20 min at 4° C.

[0099] Following the second wash, the cells were resuspended in 20 mlcold 0.05 M Na-phosphate buffer (pH 6.1). The suspensions were sonicatedon ice using a Branson Sonifier 250 at the following parameters: timer,4 cycles of each 5 min (each cycle followed by cooling to preventexcessive heating of the suspensions); output control, 2; and dutycycle, 30%).

[0100] The resulting sonicated cell material was centrifuged at 6,000rpm (Sorvall centrifuge with SS-34 rotor) for 25 min at 4° C. Thesupernatants (cell-free extracts) and pellets (cell debris) wereseparated and stored at −20° C. until enzymatic characterisation andanalysis for presence of intracellular haemin.

[0101] 1.6 Enzymatic characterisation

[0102] The enzymatic assays were all performed at 30° C. using a SecomamAnthelie spectrophotometer provided the Winelie software (ver. 1.50).The cell-free extracts were thawed on ice.

[0103] 1.6.1 Measurement of NADH oxidase activity

[0104] NADH oxidase activity of the cell-free extracts was measured bymonitoring the oxidation of NADH at 340 nm in a reaction mixture havingthe following composition: 50 mM Tris-acetate buffer (pH 6.0), 0.5 mMfructose-1,6-diphosphate and 0.5 mM NADH.

[0105] 1.6.2 Measurement of Lactate dehydrogenase activity

[0106] Lactate dehydrogenase activity of the cell-free extracts wasmeasured by monitoring the oxidation of NADH at 340 nm in a reactionmixture having the following composition: 50 mM Tris-acetate buffer (pH6.0), 0.5 mM fructose-1,6-diphosphate, 25 mM pyruvate and 0.5 mM NADH.

[0107] The lactate dehydrogenase activity was subsequently corrected forNADH oxidase activity.

[0108] One unit of enzyme activity (U) is defined as the activityrequired for oxidising 1 μmol of NADH per minute.

[0109] 1.6.3 Protein determination

[0110] For measuring the protein concentration of the cell-free extract,the Bicinchoninic acid (BCA) assay (Pierce, Rockford, USA) was used withAlbumin Standard (Pierce) as protein standard.

[0111] 1.7 Method of measuring oxygen content reducing effect

[0112] Samples collected during the fermentations were thawed andanalysed for their capacity to remove oxygen from milk (low pasteurisedskimmed milk). This oxygen reducing effect was assayed in the milk at30° C.

[0113] For each fermentation sample, the oxygen content of the milk wasmeasured regularly by a M0128 Dissolved Oxygen Meter (Mettler Toledo)following inoculation. The initial oxygen concentration of the milk was8.3 mg/kg.

[0114] 1.8 Analysis of porphyrins and cytochromes

[0115] 1.8.1 Sample preparation

[0116] Supernatant of sonicated cells

[0117] To 190 μl of the clear supernatant (cell-free extract) was added5 μl 3% hydrochloric acid to arrest enzymatic activity, and the samplewas analysed using HPLC-MS.

[0118] Pellet (cell debris)—procedure A

[0119] About 60 mg (weighed accurately) of the pellet was weighed intoan Eppendorf tube. 1 ml 88% formic acid was added and the tube waswhirly mixed. The suspension was left for 60 min at ambient temperaturein order to extract cytochromes and porphyrins from the cells. The tubewas centrifuged (6 min at 10,000 rpm using an Eppendorf centrifuge 5415)and the supernatant analysed by HPLC-MS.

[0120] Pellet (cell debris)—procedure B

[0121] About 250 mg (weighed accurately) of the pellet was weighed intoa 10 ml plastic tube. 4 ml 88% formic acid was added and the tube waswhirly mixed. The suspension was left for 60 min at ambient temperaturein order to extract cytochromes and porphyrins from the cells. Thesample was distributed into 4 Eppendorf tubes which were centrifuged for6 min at 10,000 rpm using an Eppendorf centrifuge 5415. 90 μl aliquotsof the supernatant was mixed with 10 μl standard haemin solution withvarying haemin concentrations rendering a final concentration of addedhaemin of 0; 0.2; 0.5 and 1.0 ppm, respectively. The samples were whirlymixed and analysed by HPLC-MS. The area of the specific haemin signalwas used for the quantification.

[0122] The dry-matter content of the cell debris was determined using aMettler PM480 Delta Range balance equipped with a Mettler LP-16 dryingdevice (mode: 120 sec.; calc.: %; temp.: 105° C.). Approximately 300 mgcell debris was placed on 500 mg pumice granules.

[0123] 1.8.2 HPLC-MS conditions for procedure A

[0124] 20 μl sample was injected into the HPLC-MS system consisting of aPE Series 200 HPLC provided with a Vydac C4 column (cat.#214TP51) keptat 40° C. coupled to a PE-SCIEX API150EX Mass Spectrometer (MS) with anTurbolon Spray Inlet (L=2, H=7; drying gas flow: 8 l/min and temperature300° C.). The analytes were eluted using the pump settings shown below(Table 1). TABLE 1 HPLC-MS pump settings Time [min] Flow [ml/min] A [%]B [%] 0 0.050 100 0 5 0.050 100 0 15 0.050 60 40 45 0.050 0 100 55 0.0500 100 57 0.050 100 0 61 0.050 100 0

[0125] Mobile phase A: 0.01% (v/v) trifluoroacetic acid+0.1% (v/v)acetic acid in Milli-Q-Water.

[0126] Mobile phase B: 0.008% (v/v) trifluoroacetic acid+0.1% (v/v)acetic acid in acetonitrile.

[0127] The MS signal was obtained in positive mode using differentconditions in two mass ranges:

[0128] low mass range m/z=450-650: step size was 0.250 amu, dwell time 5ms, OR=200, RNG=400

[0129] high mass range m/z=1000-1800: the step size was 1 amu, dwelltime 5 ms, OR=50, RNG=200

[0130] 1.8.3 HPL C-MS conditions for procedure B

[0131] The HPLC-MS conditions for procedure B were as described forprocedure A except for the following changes with respect to the datacollections:

[0132] low mass range m/z=450-650: step size was 0.500 amu, dwell time 5ms, pause time 5 ms

[0133] specific haemin mass m/z=616.3 m/z: the step size was 0 amu,dwell time 2000 ms, pause time 5 ms

[0134] 2. Results

[0135] 2.1 Results of the enzymatic analysis

[0136] The specific NADH oxidase and lactate dehydrogenase activities ofpellet-frozen cells harvested from each of the four fermentations arelisted in Table 2. No NADH oxidase activity was detected in cells fromthe two anaerobic fermentations A and B. Under aerobic conditions(fermentations C and D), the specific NADH oxidase activity is loweredby 25-30% by the addition of haemin to the fermentation medium.Likewise, addition of haemin lowers the specific lactate dehydrogenaseactivity by 15-20% under aerobic conditions. TABLE 2 Enzymaticactivities of the cells from the four fermentations [U/mg protein].Fermentation Fermentation Fermentation Fermentation A B C D NADH oxidase<0.01 <0.01 0.59 0.43 Lactate dehy- 19.4 18.4 11.2 9.3 drogenase

[0137] 2.2 Results for oxygen content reducing effect

[0138] The specific oxygen reducing effect of a sample of cells takenduring each of the four fermentations is shown in Table 3. As shown inTable 3, the cells which had been grown aerobically in the presence ofhaemin (fermentation D) were capable of lowering the oxygen content ofthe milk to a higher extent than cells taken from any of the three otherfermentations. TABLE 3 Oxygen content reducing effect of the cells fromthe four fermentations Fermentation Fermentation FermentationFermentation A B C D Optical density 13.2 13.9 12.8 13.3 Inoculation0.091 0.086 0.094 0.090 level [%-(w/w)] Dissolved 4.9 4.7 3.6 2.6 oxygenafter 2 hours [mg/kg] Oxygen 41 43 57 69 removed [% of initial amount]

[0139] 2.3 Results of detection of porphyrins—procedure A

[0140] Using the HPLC-MS method described above, haemin is eluting at aretention time of 33.3 min and is detected as the molecular ion at m/z616.3 (FIGS. 1a and 1 b). No haemin was detected in any of the cell-freeextracts from the four fermentations, whereas haemin was detected in thecell pellets (cell debris) when haemin had been present in thefermentation medium (FIGS. 3a, 3 b, 5 a, and 5 b). No haemin wasdetected in cell pellets (cell debris) from fermentations performed inthe absence of haemin (FIGS. 2a, 2 b, 4 a and 4 b).

[0141] 2.4 Results for detection of cytochromes

[0142] Using the HPLC-MS method described above, cytochrome c is elutingat a retention time of 23.4 min (FIGS. 6a and 6 b) and is detected asthe multiply charged intact protein (m/z 1031; 1124; 1237; 1374; 1546;1766) as well as the iron-porphyrin IX ion at m/z 617.3. The method canbe applied for detection of other cytochromes than cytochrome c.

[0143] 2.5 Quantification of porphyrins—procedure B

[0144] Using purified haemin as standard, the cellular content of haeminwas quantified for each of the fermentation A, B, C and D by HPLC-MSusing standard addition (Table 4). The dry-matter content of cell debriswas quantified to 18.5% (w/w).

[0145] As described above, no haemin was detected in cells harvestedfrom fermentations performed on a medium not supplemented with haemin(fermentations A and C), whereas 41 ppm (on a dry weight basis) ofhaemin was detected in cells from fermentations performed on a mediumsupplemented with haemin (fermentations B and D). TABLE 4 Quantificationof cellular haemin Fermentation Fermentation Fermentation Fermentation AB C D Haemin in wet 0 7.6 0 7.6 cell debris [ppm] Haemin in dry 0 41 041 cell debris [ppm]

Experiment 2

[0146] 1. Materials and methods

[0147] 1.1 Microorganisms

[0148] A mixed strain starter culture of Lactococcus lactis strains wasapplied in this experiment.

[0149] 1.2 Medium composition

[0150] Fermentation E was likewise performed using a conventionalcomplex fermentation medium containing lactose as the carbon source.Haemin was added to a final concentration of 10 mg/l.

[0151] 1.3 Fermentation conditions

[0152] The fermentation was inoculated with a concentrated cellsuspension of the Lactococcus lactis culture. Air was sparged throughthe fermentation broth at a rate sufficient to maintain the dissolvedoxygen concentration above 50% of saturation level. The fermentation wasrun at a temperature of 30° C. The culture was allowed to acidify to pH6.2, whereafter pH was maintained at 6.2 by controlled addition of 13.4N NH4OH.

[0153] 1.4 Downstream processing

[0154] The downstream processing was as described in Experiment 1.

[0155] 1.4.1 Determination of dry-matter content of frozen pellets

[0156] About 5 g of frozen pellets were weighed into a metal traypositioned on a Sartorius MA 30 balance equipped with a heating deviceand heated at 160° C. until constant weight. From the loss of weight ofthe sample, the dry-matter content was calculated.

[0157] 1.5 Sample preparation

[0158] About 20 g of frozen pellets from fermentation E was weighed outaccurately. These pellets were washed twice in cold 20 mM sodiummorpholinic propane sulfonic acid (MOPS) buffer (pH 7.4). Following eachstep of washing, the suspension was centrifuged at 4° C. on a Sorvall RC50 Plus centrifuge with SS-34 rotor (following first wash: 5,000 rpm for20 min.; following second wash: 8,000 rpm for 30 min.).

[0159] Following the second wash, the cells were resuspended in 10 mlcold MOPS buffer (20 mM, pH 7.4) containing 0.5 mMphenylmethylsulfonylflouride and 5 mM MgSO4 as described in Winstedt etal. (2000). The suspension was then passed through a French Press fivetimes and subsequently centrifuged on the Sorvall centrifuge (4° C.,4,000 rpm, 30 min.). The supernatant was separated and stored at −20° C.until further analysis.

[0160] 1.6 Spectrophotometric detection and quantification ofcytochromes

[0161] Prior to analysis, the frozen supernatant was thawed on ice andcentrifuged on the Sorvall centrifuge (4° C., 4,000 rpm, 30 min.).

[0162] 1.5 ml plastic cuvettes were used. 1 ml supernatant was used forboth cuvettes. When stated, cyanide was added as 10 μl 0.5 M KCNresulting in a concentration of 5 mM. Also when stated, sodiumdithionite was added directly in dry form to cuvettes for reducing thesamples.

[0163] A Shimadzu UVPC 2101 spectrophotometer was applied (1 nm step; 5nm slit; “very slow” speed).

[0164] 1.6.1 CN-Ox and CN/Red-CN spectra

[0165] Untreated sample (=oxidised) was entered into the referencecuvette as well as into the measurement cuvette. Identity of the samplesin the two cuvettes was ensured (Ox-Ox spectrum). KCN was added to themeasurement cuvette, and the spectrum was recorded (CN-Ox spectrum).Subsequently, KCN was added to the reference cuvette and identity of thesamples in the two cuvettes was ensured (CN-CN spectrum). Finally,sodium dithionite was added to the measurement cuvette and the spectrumwas recorded after 5-10 min. (CN/Red-CN spectrum).

[0166] 1.6.2 Red-Ox spectrum

[0167] Untreated sample (=oxidised) was entered into the referencecuvette as well as into the measurement cuvette. Identity of the samplesin the two cuvettes was ensured (Ox-Ox spectrum). Then, sodiumdithionite was added to the measurement cuvette and the spectrum wasrecorded after 5-10 min. (Red-Ox spectrum).

[0168] 1.6.3 Treatment of spectra

[0169] The recorded difference spectra were subjected to minortransformations prior to further data analysis. A straight line wassubtracted from the spectra in order to obtain a spectrum in the 500 to700 nm range without too much tilting. The baseline for the peak at 630nm was calculated from the values at 612 nm and 658 nm.

[0170] 2. Results

[0171] The recorded difference spectra for cells from fermentation E areshown in FIG. 7. The CN-Ox spectrum (thin line) is without anycharacteristics in the 500 to 700 nm range. The CN/Red-CN spectrum(dashed line) exhibit a peak at 630 nm—a peak that is smaller than thecorresponding peak on the Red-Ox spectrum (thick line), i.e. cyanide“protects” the cytochrome from reduction with dithionite. Theseobservations together with the trough at 650 nm is a strong indicationthat the cytochrome is cytochrome d (Gil et al., 1992).

[0172] The height of the 630 nm peak is 0.0008 AU. By applying anextinction coefficient of 18.8 mM−1 cm−1 (Kita et al., 1984), thiscorresponds to a cytochrome d concentration of 0.04 μM. Assuming amolecular weight of around 100 kDa, the concentration of cytochrome d is4 mg/l. Since the initial 20 grams of frozen pellets from fermentation Econtained 15.7% (w/w) of dry-matter, the cellular cytochrome dconcentration is 13 ppm (mg per kg dry-matter).

[0173] No cytochromes were detected in frozen pellets from the anaerobicfermentation in the absence of haemin (fermentation A).

REFERENCES

[0174] Gil, A., Kroll, R. G. & Poole, R. K. 1992. The cytochromecomposition of the meat spoilage bacterium Brochothrix thermosphacta:identification of cytochrome a3- and d-type terminal oxidases undervarious conditions. Archives of Microbiology 158:226-233.

[0175] Kaneko, T., Takahashi, M. & Suzuki, H., 1990, Acetoinfermentation by citrate-positive Lactococcus lactis subsp. lactis 3022grown aerobically in the presence of hemin or Cu²⁺, Applied andEnvironmental Microbiology 56:2644-2649.

[0176] Kita, K., Konishi, K. & Anraku, Y. 1984. Terminal oxidases ofEscherichia coli aerobic respiratory chain. Journal of BiologicalChemistry 259:3375-3381.

[0177] Ritchey, T. W. & Seeley Jr., H. W., 1976, Distribution ofcytochrome-like respiration in Streptococci. Journal of GeneralMicrobiology 93:195-203.

[0178] Sijpesteijn, A. K., 1970, Induction of cytochrome formation andstimulation of oxidative dissimilation by hemin in Streptococcus lacticand Leuconostoc mesenteroides. Antonie van Leeuwenhoek 36:335-348.

[0179] von Wachenfeldt, C. & Hederstedt, L. 1992. Molecular biology ofBacillus subtilis cytochromes. FEMS Microbiology Letters 100:91-100.

[0180] Winstedt, L., Frankenberg, L., Hederstedt, L. & von Wachenfeldt,C. 2000. Enterococcus faecalis V583 contains a cytochrome bd-typerespiratory oxidase. Journal of Bacteriology 182:3863-3866.

1. A culturally modified lactic acid bacterial cell that has, relativeto the cell from which it is derived, an increased content of aporphyrin compound.
 2. A cell according to claim 1 that contains atleast 0.1 ppm on a dry matter basis of a porphyrin compound.
 3. A cellaccording to claim 1 that contains a detectable amount of a cytochrome.4. A cell according to claim 3 that contains at least 0.1 ppm on a drymatter basis of a cytochrome.
 5. A cell according to claim 4 thatcontains at least 0.1 ppm on a dry matter basis of cytochrome d
 6. Acell according to claim 1 which is of a bacterial species selected fromthe group consisting of Lactococcus spp., Lactobacillus spp.,Leuconostoc spp., Pediococcus spp., Streptococcus spp.,Propionibacterium spp., Bifidobacterium spp. and Oenococcus spp.
 7. Acell according to claim 6 where the bacterial species is of Lactococcuslactis, including Lactococcus lactis strain CHCC373 deposited under theaccession number DSM12015.
 8. A cell according to claim 1 which, when itin the form of a cell suspension is inoculated in a concentration of 10⁷cells/ml into low pasteurised skimmed milk having 8 ppm of dissolvedoxygen and leaving the milk to stand for about two hours at atemperature of about 30° C. consumes at least 25% of the oxygen.
 9. Acell according to claim 8 where the cell consumes at least 50% of thedissolved oxygen.
 10. A cell according to claim 1 , which, relative tothe cell from which it is derived, has a decreased NOX activity and/or adecreased LDH activity.
 11. A cell according to claim 10 that has a NOXactivity which is decreased by at least 10%.
 12. A cell according toclaim 10 that has a LDH activity which is decreased by at least 10%. 13.A starter culture composition comprising the culturally modified lacticacid bacterial cell of any of claims 1-12.
 14. A composition accordingto claim 13 where the composition is in the form of a frozen, liquid orfreeze-dried composition.
 15. A composition according to claim 13containing an amount of viable culturally modified lactic acid bacterialcells which is in the range of 10⁴ to 10¹² CFU per g.
 16. A compositionaccording to claim 13 that comprises cells of two or more differentlactic acid bacterial strains.
 17. A composition according to claim 13which further comprises at least one component enhancing the viabilityof the bacterial cell during storage, including a bacterial nutrientand/or a cryoprotectant.
 18. A method of reducing the oxygen content ina food or feed product or in a food or feed product starting materialcomprising adding to the product or to the starting material aneffective amount of the culturally modified lactic acid bacterial cellsaccording to any of claims 1-12 or the starter culture compositionaccording to any of claims 13-17.
 19. A method according to claim 18wherein the amount of modified cell is in the range of 10⁴ to 10¹² CFUper g.
 20. A method according to claim 18 wherein the starting materialfor the food product is selected from the group consisting of milk, avegetable material, a meat product, a fruit juice, a must, a wine, adough and a batter.
 21. A method of improving the shelf life and/or thequality of an edible product comprising adding to the product aneffective amount of the culturally modified lactic acid bacterial cellsaccording to any of claims 1-12 or the starter culture compositionaccording to any of claims 13-17.
 22. A method of preparing a fermentedfood or feed product, comprising adding an effective amount of theculturally modified lactic acid bacterial cell according to any ofclaims 1-12 or the composition of any of claims 13-17 to a food or feedproduct starting material, wherein the cell or the composition iscapable of fermenting said starting material to obtain the fermentedfood or feed.
 23. A method according to claim 22 wherein the startingmaterial for the food product is selected from the group consisting ofmilk, a vegetable material, a meat product, a fruit juice, a must, awine, a dough and a batter.
 24. A method according to claim 23 whereinthe resulting fermented food product is a dairy product including aproduct selected from the group consisting of cheese and buttermilk. 25.Use of the lactic acid bacterial cell of any of claims 1-12 or thecomposition of any of claims 13-17 for the production of a metaboliteproduced by the cell or the composition or by a non-modified cellco-cultivated therewith.
 26. Use according to claim 25 where themetabolite is selected from the group consisting of lactic acid,acetaldehyde, α-acetolactate, acetoin, acetate, ethanol, diacetyl and2,3-butylene glycol.
 27. Use of the lactic acid bacterial cell of any ofclaims 1-12 or the composition of any of claims 13-17 for the productionof a bacteriocin.
 28. Use according to claim 27 where the bacteriocin isselected from the group consisting of nisin, reuterin and pediocin.